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Chemical Composition, Antioxidant and Antimicrobial Activities of the Essential Oils of Three Algerian Lamiaceae Species Khadidja Houda Benabed, Nadhir Gourine, Mohamed Ouinten, Isabelle Bombarda, Mohamed Yousfi

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Khadidja Houda Benabed, Nadhir Gourine, Mohamed Ouinten, Isabelle Bombarda, Mohamed Yousfi. Chemical Composition, Antioxidant and Antimicrobial Activities of the Essential Oils of Three Alge- rian Lamiaceae Species. Current Nutrition and Food Science, Bentham Science Publishers, 2017, 13 (2), pp.97 - 109. 10.2174/1573401313666170104105521. hal-01777681

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The user has requested enhancement of the downloaded file. Send Orders for Reprints to [email protected] Current Nutrition & Food Science, 2017, 13, 1-13 1 RESEARCH ARTICLE Chemical Composition, Antioxidant and Antimicrobial Activities of the Essential Oils of Three Algerian Lamiaceae Species

Khadidja Houda Benabeda, Nadhir Gourinea,*, Mohamed Ouintena, Isabelle Bombardab and Mohamed Yousfia aLaboratoire des Sciences Fondamentales LSF, Université Amar Télidji, Laghouat, BP. 37G, Laghouat, Algérie; bLabo- ratoire LISA, Équipe METICA, EA 4672, Université Aix-Marseille, Marseille, France

Abstract: Background: The present work investigates the chemical composition, the antioxidant and the antimicrobial activities of the Essential Oils (EOs) of three species of the Lamiaceae family gowning in Algeria: Thymus vulgaris L., Thymus algeriensis Boiss. & Reut. and Mentha pulegium L. Methods: Essential Oils (EOs) obtained by hydrodistillation of the aerial parts of the studied plants were analyzed by GC and GC-MS. The antioxidant activity of the EOs was determined using two different assays: free radical scavenging activity of DPPH• (2,2-diphenyl-1-picrylhydrazyl) and Phosphomolybdenum reducing power. The EOs were also tested for their antibacterial and antifungal activities against eight pathogenic bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Sal- A R T I C L E H I S T O R Y monella typhi, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Methicillin resistant strain Received: September 03, 2016 of S. aureus “MRSA” and Enterococcus faecalis); a yeast (Candida albicans); and a fungi Revised: December 20, 2016 Accepted: December 30, 2016 (Fusarium oxysporum).

DOI: Results: For DPPH assay, T. vulgaris presented very interesting activity. At the opposite, T. al- 10.2174/157340131366616120 geriensis (Aflou) and M. pulegium, were the most active EOs in term of Phosphomolybdenum assay. 9162354 The antimicrobial activity of T. vulgaris was found to be the most active EO and exhibited important resistance against most of studied bacteria. For disc diffusion test, the most active EO plant was T. algeriensis. Alternatively, and for antifungal activity, T. vulgaris presented the highest value of MFC. Conclusion: The antioxidant activity test’s results showed that the EOs exhibited important reducing powers but weak scavenging activities. On the other hand, it was found that some EO samples have shown very interesting antimicrobial activities. Actually, among the investigated EOs, T. vulgaris presented the strongest antibacterial and antifungal activities.

Keywords: Thymus vulgaris L., Thymus algeriensis Boiss. & Reut., Mentha pulegium L., DPPH assay, phosphomolybdenum assay, antimicrobial activity, essential oil.

1. INTRODUCTION natural products such as antioxidants is increasingly important. Thus, various sources of antioxidants of plant origin To cure its ills, man had always relied on animals and have been studied in recent years [3]. plants. These have always been the basis of the traditional systems of medicine for thousands of years and continue to The use of essential oils as functional ingredients in date to provide new remedies [1]. Recently, the introduction foods, drinks, toiletries and cosmetics is gaining momentum of traditional medicine, as an alternative form, as well as the [4]. Additionally, the use of essential oils is becoming popu- development of microbial resistance to antibiotics has led lar to increase the shelf-life of food products, since consum- researchers to study the antimicrobial activity of medicinal ers are more conscious about the health problems caused by plants [2]. On the other hand, the different synthetic antioxi- several synthetic preservatives [5, 6]. dants used in food, were suspected to have negative health The purpose of this research was to investigate, firstly the effects. It is for this reason that the interest for the study of chemical composition and the antioxidant properties of three EOs (T. vulgaris L., T. algeriensis Boiss. & Reut. and M. pulegium L.) collected from two regions in the center of Al- *Address correspondence to this author at the Laboratoire des Sciences geria; and secondly, to examine their antimicrobial activities Fondamentales LSF, Université Amar Télidji, Laghouat, BP. 37G, Laghouat, Algérie; E-mails: [email protected] or against eight pathogenic bacteria (Klebsiella pneumoniae, [email protected] Pseudomonas aeruginosa, Salmonella typhi, Escherichia

1573-4013/17 $58.00+.00 © 2017 Bentham Science Publishers 2 Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al. coli, Bacillus cereus, Staphylococcus aureus, Methicillin Mentha pulegium L. is one of the Mentha species com- resistant strain of S. aureus “MRSA” and Enterococcus fae- monly known as pennyroyal. It is native species of Europe, calis); a yeast (Candida albicans); and a fungi (Fusarium North Africa and in Asia Minor and near East. The flowering oxysporum). aerial parts of M. pulegium L. has been traditionally used for Only very few reports on the determination of the anti- its antiseptic for treatment of cold, sinusitis, cholera, food poisoning, bronchitis and tuberculosis, and also as antiflatu- oxidant activity of the essential oil using Phosphomolybde- lent, carminative, expectorant, diuretic, antitussive, menstru- num assay and applied for of T. vulgaris were found in lit- ate. Some pharmacological effect of M. pulegium L. essential erature [7-9]. For these reports, the antioxidant values are oil such as abortifacient effect in rat myometrium, cytotoxic difficult to use for comparison purpose since different meth- activity against different human cell lines and its antioxidant ods of expressions or different antioxidant of reference were engaged. In the same context, and as best of our knowledge, effect were confirmed [19]. there are no reports dealing with the Phosphomolybdenum In Algeria it is much appreciated, so much so that it is assay of the EO for T. algeriensis Boiss. & Reut. and M. used to prepare a traditional dish: the potato stew with the pulegium L. As far as we know, this is the first report that pennyroyal "Batata Fliou". Its pleasant odor seems to dis- investigates the antioxidant antimicrobial activities of the please some kind of parasites, and its insecticidal power is essential oils of the plants from these regions of collection well established. Formerly, it was burned in fleece infested (Djelfa and Aflou “Laghouat”) from Algeria. Furthermore, premises. It was also used as a lotion on the coat of domestic we have used for the first time a special form of Fusarium animals to rid them of their parasitic pests [16]. oxysporum which was isolated from a fragment of date palm from Ghardaia region to investigate the antifungal activity. 2.2. Plants Materials Collections In addition, the antioxidant activity of the EOs determined by DPPH and Phosphomolybdenum assays were examined The study was carried out using four EO samples ob- for the first time for these regions. Finally, correlations be- tained from three species belonging to the Lamiaceae family: tween chemical compositions of the EOs and their antioxi- M. pulegium L. (Djelfa), T. vulgaris L. (Djelfa) and T. al- dant and antimicrobial activities were investigated using geriensis Boiss. & Reut. (from two different stations: Djelfa statistical analysis; this was done in order to determine the and Aflou-Laghouat). Since T. algeriensis, wide spreads nature of the chemical component(s) responsible(s) for the over a larger region, we have picked up two samples from different activities of the EOs. different locations in order to compare theirs EOs composition differences as well as theirs antioxidant and antibacterial activities behaviors. The different plant samples were col- 2. MATERIALS AND METHODS lected in 2010 during the flowering stage; (end of May for 2.1. Plants Descriptions M. pulegium and T. algeriensis, and the beginning June for T. vulgaris). Voucher specimens (TV/06/10, TA-AFL/06/10, Thymus vulgaris L. is a perennial herb indigenous in TA-DJF/06/10, MP/06/10) were deposited in the herbarium central and southern Europe, Africa and Asia. It is widely of the Fundamental Sciences Research Laboratory at Lag- used in folk medicine in the treatments of variety of diseases houat University. such as gastroenteric and bronchopulmonary disorders, as well as due to its anthelmintic, carminative, sedative and 2.3. Essential Oil Extraction diaphoretic properties [10]. It has been reported that its EO possesses numerous biological activities including antiworm, The different samples of EOs were obtained by hydrodis- antiseptic, antispasmodic, antimicrobial [11] and antioxidant tillation using a Clevenger type apparatus; subsequently the [12]. In addition, it is a well-known species of the genus obtained EOs were dried over anhydrous sodium sulfate, and Thymus, extensively studied for its chemical and biological stored in a dark at 4 °C. activities [13-15]. 2.4. Essential Oil Analysis In Algeria, T. vulgaris is one of the most useful popular remedies in the treatment of respiratory affections (colds, flu, The GC analysis was performed using a gas chromato- angina) and gastric disorders (dyspepsia, cramps) [16]. graph type Chrompack CP 9002, equipped with a fused silica capillary column DB-5 (30m0.32mm, 0.25m film thick- Thymus algeriensis Boiss. & Reut. is the most wide- ness) and Flame Ionization Detector (FID). The carrier gas spread North African species, endemic to Libya, Tunisia, was nitrogen at a flow rate of 1mL/min. The column tem- Algeria and Marocco. Fresh or dried, it is largely used only perature was programmed from 50 °C (3 minutes) to 250 °C as a culinary herb. T. algeriensis is also used in traditional at 2 °C/min, and then maintained at 250 °C for 10 min. The medicine, as a fresh or dry seasoning, in respiratory and di- temperatures of the injector and detector were set at 250 °C. gestive tube disorders and against abortion [17]. Volumes of 1L of diluted samples (1:100 v/v, in ethanol) In Algeria, T. algeriensis is used as a treatment for stom- were injected manually using split less mode. Linear reten- achic, diaphoretic, antispasmodic and stimulating. The sum- tion indices of the components were calculated using a series mits and the young flowering twigs are used. Infusion is use- of n-alkane (C9-C25) analyzed under the same operating con- ful against all infectious diseases, such as influenza, pneu- ditions as those of the EOs samples. monia and respiratory tract diseases. It is used in friction in The GC/MS analysis was performed on an AGILENT cases of neuralgia and sciatica and as odontalgic on decayed 6890 GC/CMSD 5973 equipped with a capillary column toothache [18]. UB-Wax (30m0.25mm, 0.25m film thickness) and a 70 Chemical Composition, Antioxidant and Antimicrobial Activities Current Nutrition & Food Science, 2017, Vol. 13, No. 0 3 eV EI Quadruapole detector. Helium was the carrier gas, at a 2.8. Antimicrobial Tests flow rate of 1mL/min. Injector and MS transfer line tempera- The antimicrobial activity of the different EO samples tures were set at 250 °C and 220 °C, respectively. Column was screened by the disk diffusion test [23], and then the temperature was programmed same as gas chromatography. minimal inhibitory and bactericidal concentrations were de- Diluted samples (1:100 v/v, in ethanol) of 1L were also injected manually using split less mode. termined using a dilution test [24]. The antifungal activity was tested using a direct contact test as described by El Ajjouri et al. [25]. The EOs constituents were identified by comparison of their linear retention indices and mass spectra with those in 3. RESULTS the computer library (NIST MS Library) and with literature 3.1. Essential Oil Yield and Chemical Composition data [20]. The yields of the studied EOs were varying from moder- 2.5. DPPH Assay (Free Radical Scavenging Activity) ate to low values. They were somehow important for M. pulegium and T. vulgaris. In the other hand, they were rang- For this test, the ability of hydrogen donating or radical ing from low to poor values for T. algeriensis (Table 1). scavenging was measured using the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) [21]. The EOs dilutions The chemical compositions of the different EO samples were prepared in absolute ethanol, and then 1mL of each are presented in Table 1. For the EO of T. vulgaris, thirty dilution was added to 1mL of a 200M DPPH solution. The two different compounds representing 96.47% of the total oil mixtures were incubated in dark for 30 min; afterwards the were identified, with proportions of monoterpene hydrocar- absorbance was measured at 517 nm against a blank. The bons and oxygenated monoterpenes equal to 52.98% and inhibition percentage was calculated using the following 39.14%, respectively. The major component of this oil was formula: the -terpinene (25.70%) followed by thymol (20.83%), then p-cymene (20.04%). The oxygenated compounds were also A A I(%) = 0 100 present in lower amounts than the hydrocarbons: thymol A methyl ether (7.82%), linalool (4.76%) and borneol (2.54%). 0 The -caryophyllene (2.28%) was the main sesquiterpene Where: compound for this EO. A0 is the absorbance of the control reaction (containing all For the EO of pennyroyal (M. pulegium), twenty eight reagents except the test compound) and A is the absorbance compounds were identified representing 76.38% of the total of the test compound. EO. The oil was mainly composed of pulegone (54.92%), which accounts for almost the majority of oxygenated 2.6. Phosphomolybdenum Assay monoterpenes. Monoterpene hydrocarbons represented only This assay is based on the reduction of Molybdate (VI) to a small fraction of the EO content (2.67%). Molybdate (V) by the antioxidant compounds and the forma- For the case of T. algeriensis, forty four components tion of a green phosphate/Molybdate (V) complex at acidic were identified for the sample of Djelfa and thirty three pH with a maximum absorbance at 695 nm [22]. The EOs components of that of Aflou (Laghouat), both accounting for samples were diluted in absolute ethanol. An amount of 200 83.30 and 81.06% of the total EOs, respectively. Camphor L of each dilution was mixed with 2 mL of reagent (0.6 M was the predominant compound in the sample of Aflou sulfuric acid, 28 mM sodium phosphate, and 4 mM ammo- (Laghouat) with a percentage of 17.68%, this content agrees nium molybdate). The mixture was then incubated in a water with the results of Amarti et al. [26] and Zouari et al. [27] bath set at 70 °C for 90 min [22]. The absorbance was de- who have detected camphor as major compound for T. al- termined at 695 nm. The results were expressed in terms of ascorbic acid equivalent AEAC. geriensis from Morocco and Tunisia, respectively. The sample of Djelfa was predominantly composed of -terpinenyle acetate (27.32%), a compound completely absent in the sam- 2.7. Microbial Strains ple of Aflou. Camphor was also present, but with a lower The different samples of EOs were tested against eight content (10.77%) than recorded for the sample of Aflou bacteria: Klebsiella pneumoniae ATCC 700603, Pseudo- (Laghouat). The eucalyptol was one of the major compounds monas aeruginosa ATCC 27853, Salmonella typhi (isolate), in the EO sample of Aflou (10.04%), whereas it represented Escherichia coli ATCC 25922, Staphylococcus aureus only 2.47% for the sample of Djelfa. The EO of Aflou sam- ATCC 25923, Methicillin resistant strain of S. aureus ATCC ple showed a high content of monoterpene hydrocarbons 43300, Bacillus cereus ATCC 11779, Enterococcus faecalis (41.29%) compared to that of Djelfa (13.71%), with a pre- ATCC 29212 ; a yeast: Candida albicans (isolate) and a fil- dominance of camphene (8.73%), -pinene (7.84%) and amentous fungi Fusarium oxysporum f. sp. albedinis. The myrcene (6.95%). On the other hand, the two samples fungal species Fusarium oxysporum special form albedinis showed almost equal percentages of borneol (5.58 and was isolated from a fragment of date palm provided by the 5.68%). Sesquiterpenes were minor compounds in the EOs Regional Station of Plant Protection of Ghardaia (Algeria). of the two regions with a total percentage of 5.46 and 3.04% The culture was then purified by performing a monospore for Djelfa and Aflou respectively. culture. 4 Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al.

Table 1. Chemical composition of the essential oil samples: Thymus vulgaris, Thymus algeriensis and Mentha pulegium.

Thymus vulgaris Thymus algeriensis Mentha pulegium Components LRIa LRIb (Djelfa) (Djelfa) (Djelfa) (Aflou-Laghouat)

-Thujene 921 924 1.10 0.05 0.26 0.05

-Pinene 926 932 0.92 3.58 7.84 0.06

Camphene 938 946 1.08 2.75 8.73 0.02

Sabinene 961 969 0.04 0.37 – 0.09

-Pinene 964 974 0.19 0.13 1.43 0.40

Myrcene 984 988 0.99 1.34 6.95 –

-Phellandrene 995 1002 0.19 0.01 0.02 0.01

-Carene 1002 1008 0.07 0.01 – 0.04

-Terpinene 1008 1014 1.93 0.20 – –

p-Cymene 1014 1020 20.04 0.97 0.69 –

Limonene 1019 1024 0.17 1.11 – 0.75

Eucalyptol 1021 1059 0.40 2.47 10.04 0.98

(Z)--Ocimene 1027 1032 – 0.03 5.23 –

-Terpinene 1052 1054 25.70 0.32 – 0.27

Terpinolene 1077 1086 0.16 0.37 0.10 –

Linalool 1088 1095 4.76 1.46 1.34 0.43

-Thujone 1103 1112 0.07 0.47 0.35 –

allo-Ocimène 1118 1128 – 0.02 – 0.05

Camphor 1123 1141 0.03 10.77 17.68 –

neo-isopulegol 1134 1167 – - - 1.02

Pinocarvone 1135 1160 – 2.99 1.65 –

Isopulegol 1142 1145 – – – 0.10

Borneol 1149 1165 2.54 5.58 5.68 1.06

Terpinen-4-ol 1162 1174 0.70 2.56 – 0.27

-terpineol 1174 1186 0.28 3.12 1.25 –

Naphtalene 1186 1178 0.08 1.71 1.02 0.11

Thymol methyl ether 1218 1232 7.82 – – –

Nerol 1219 1227 – 0.63 0.96 –

Pulegone 1224 1233 – – – 54.92

Carvone 1234 1239 – 0.17 0.02 3.32

Linalyl acetate 1242 1248c – 0.33 – 1.71

iso-Pulegyl acetate 1264 1275 – – – 6.50

Bornyl acetate 1268 1284 – 4.84 0.50 1.05

Thymol 1279 1289 20.83 0.03 0.04 –

Carvacrol 1294 1298 2.03 0.02 0.03 –

Eugenol 1330 1356 – – – 0.22 Chemical Composition, Antioxidant and Antimicrobial Activities Current Nutrition & Food Science, 2017, Vol. 13, No. 0 5

Table 1. contd…

Thymus vulgaris Thymus algeriensis Mentha pulegium Components LRIa LRIb (Djelfa) (Djelfa) (Djelfa) (Aflou-Laghouat)

-Terpinenyl acetate 1341 1346 – 27.32 – –

Neryl acetate 1350 1359 – 1.69 5.76 0.20

Geranyl acetate 1364 1379 – 0.42 0.45 –

-Caryophyllene 1408 1417 2.28 0.12 0.22 1.78

Aromadendrene 1425 1439 0.10 0.16 0.05 –

allo-Aromadendrene 1445 1458 0.05 0.07 0.11 –

Germacrene D 1466 1484 0.03 0.25 0.01 0.57

Germacrene A 1496 1508 0.90 0.11 0.51 –

-Cadinene 1504 1522 – 0.08 – –

(Z)-Nerolidol 1517 1531 – 0.04 0.05 –

Germacrene B 1544 1559 – 0.18 0.81 –

Ledol 1553 1602 0.30 2.72 0.42 –

Spathulenol 1555 1577 0.29 0.77 – –

Caryophyllene oxide 1559 1582 0.40 0.96 0.86 0.40

Total Identified (%) 96.47 83.30 81.06 76.38

Monoterpene hydrocarbons 52.98 13.71 41.29 2.67

Oxygen-containing monoterpenes 39.14 64.13 36.73 70.96

Sesquiterpene Hydrocarbons 3.36 0.79 0.90 2.35

Oxygen-Containing Sesquiterpenes 0.99 4.67 2.14 0.40

Essential oil yield (% m/m) 1.34 0.56 0.27 1.47 a LRI: Linear Retention Indices determined experimentally on apolar column DB-5. b LRI: Linear Retention Indices obtained from literature [20]. c LRI: Linear Retention Indices obtained from literature [61].

Table 2. Antioxidant acitivity of the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium using DPPH free radical scavenging.

Essential Oil Sample EC50 (mg/mL)

Thymus vulgaris 2.3 ± 0.1

Thymus algeriensis (Djelfa) 10.2 ± 0.9

Thymus algeriensis (Aflou) > 45.0

Mentha pulegium 27.7± 2.4

Vitamin E 0.0207 ± 0.0007

Ascorbic acid 0.0078 ± 0.0001

Butylhydroxyanisole (BHA) 0.0011 ± 0.0003

3.2. Antioxidant Activity measures the ability of the sample to provide protons, whereas the phosphomolybdenum assay measures the capac- 3.2.1. DPPH Assay ity of the sample to donate electrons. Antioxidant activities of the different EO samples were The results of the tests of DPPH radical scavenging evaluated using two complementary methods: the DPPH activities of the different samples on the EOs are shown in assay, and the phosphomolybdenum assay. The DPPH test Table 2. These results are expressed as EC50 values, that are 6 Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al.

Table 3. Antioxidant acitivity of the essential oil samples of antimicrobial activities with those of literature “Tables 4(c) Thymus vulgaris, Thymus algeriensis and Mentha and 5(b)” are not evident since the evaluations conditions are pulegium using Phosphomolybdenum assay. somehow different. T. vulgaris EO sample had the strongest activity with the Essential Oil AEACa (mg/mL) highest inhibition zone diameters, while the EOs of T. algeriensis and M. pulegium showed moderate activities. Thymus vulgaris (Djelfa) 0.107 ± 0.007 In a diffusion test, the inhibition zone diameter for a Thymus algeriensis (Djelfa) 0.220 ± 0.022 component is determined by its antimicrobial activity, its Thymus algeriensis (Aflou) 0.148 ± 0.003 solubility and capacity of diffusion in the media and by the characteristics of the microorganism itself [31]. Diffusion Mentha pulegium (Djelfa) 0.159 ± 0.002 methods are likely appreciated because their simplicity and Vitamin E 0.572 ± 0.023 low cost, but these methods are not always reliable for the evaluation of antimicrobial activities of plant extracts, be- aAntioxidant reducing power is expressed as AEAC (Ascorbic acid Equivalent Antioxi- cause the absence of inhibition zone doesn’t mean that the dant Power). tested compound is inactive, especially for compounds with defined as the concentration of substrate that causes 50% low polarity that diffuse slowly in the culture media [32]. loss of the concentration of the free radicals DPPH initially 3.3.2. Minimal Inhibitory Concentration (MIC) and Bacte- introduced; the higher the antioxidant activity, the lower is ricidal Concentrations (MBC) Tests the value of EC50 [21]. In comparison with antioxidants of reference, the EO samples showed weak DPPH-radical- The determined MIC values were ranged from 0.127 scavenging activities. Their activities can be classified as mg/mL (T. vulgaris EO for B. cereus and E. faecalis) to follows: T. vulgaris>T. algeriensis (Djelfa)>M. pulegium>T. higher than 4.5 mg/mL (for Candida albicans for the two algeriensis (Aflou-Laghouat). More effectively, T. vulgaris samples of T. algeriensis). The same resistance was observed for P. aeruginosa towards all the EO samples (Table 5). presented a very interesting EC50 value, and it could be con- sidered active in comparison with the majority of common T. vulgaris EO showed MICs values between 0.12 EOs. mg/mL (for the strains B. cereus and E. faecalis) and 1.01 3.2.2. Phosphomolybdenum Assay mg/mL (for K. pneumoniae and S. aureus). The growth of MRSA, S. typhi and E. coli was inhibited at a concentration The second test named Phosphomolybdenum test, meas- equal to 0.5 mg/mL. The resistance of P. aeruginosa was ures the reducing power capacity in aqueous medium. Anti- observed even with a concentration of 2.03 mg/mL. oxidant reducing power is expressed as AEAC: Ascorbic Imelouane et al. [33] found for this same species EO MIC acid Equivalent Antioxidant Power. By definition, AEAC is values equal to 1.33 mg/mL for the strains E. coli, S. aureus defined as the concentration of an antioxidant of reference and S. epidermidis. Kaloustian et al. [34] found MICs equal (ascorbic acid in this case) in mg/mL, which gives the to 1 and 2 mg/mL for the strains E. coli and S. aureus respec- equivalent antioxidant power for a concentration of 1 mg/mL tively, while testing the EOs of T. vulgaris and T. zygis. of the EO. High value of AEAC indicates high antioxidant capacity. For the MBCs, B. cereus was the most sensitive strain, with MBC equal to 0.5 mg/mL. MRSA, S. typhi, E. coli, S. In comparison with vitamin E, the EOs showed moderate aureus and E. faecalis were inhibited at an oil concentration antioxidant capacities (Table 3). The highest antioxidant of 1.01 mg/mL. Many previous studies confirm the antimi- capacity was observed for the EO of T. algeriensis (Djelfa); crobial properties of EOs from Thymus genus [35-41], that is while the EO with the lowest activity was recorded for T. in correlation with our results. T. algeriensis EO showed a vulgaris. As a matter of fact, the EOs samples presented moderate to weak activity with more effectiveness on Gram practically close values of antioxidant activities, and they positive bacteria (Table 6), this weak activity has been also were approximately 2 to 5 times lower than vitamin E; observed by Hazzit et al. [42] for two samples of T. al- whereas, vitamin E presented almost two times lower antioxidant power than vitamin C. geriensis. 3.4. Antifungal Activity 3.3. Antimicrobial Activity Inhibition growth percentages observed for the different 3.3.1. Disc Diffusion Test EO samples against the filamentous fungi Fusarium ox- The antibacterial activities of the EOs were also investi- ysporum sp.f. albedinis are shown in Table 6. All the EO gated. Inhibition zone diameters are includes in Table 4 for tested exhibited inhibition effects against the tested fungi. the four EO samples (values are average of three repetitions). The EO of T. vulgaris showed the strongest activity with a The results showed a sensitivity of the microorganisms to- fungistatic and fungicide concentration equal to 1.753 wards all the EOs, with a remarquable resistance for Pseu- mg/mL, and 3.438 mg/mL, respectively. While the EOs of T. domonas aeruginosa. This resistance was observed by Co- algeriensis (Djelfa) and M. pulegium showed a moderate sentino et al. [28] for the EOs of T. capitatus and T. herba activity with MICs values equal to 8.963 mg/mL and 8.781 barona, Bouhdid et al. [29] for three species of T. genus and mg/mL, respectively. In another hand, these latest EOs did Hussain et al. [30] for six plants within the Lamiaceae fam- not exhibit any measured MFCs activity in the range of the ily. Let’s mention that comparisons of results of the founded concentration studied. These observed differences are proba- Chemical Composition, Antioxidant and Antimicrobial Activities Current Nutrition & Food Science, 2017, Vol. 13, No. 0 7

Table 4a. Antimicrobial activity of essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium by disc- diffusion diameter inhibition zones.

Thymus algeriensis Essential Oil Plant Thymus vulgaris Mentha pulegium Djelfa Aflou

Essential oil concentration (mg/disc) 2.23 2.32 2.23 2.27 Inhibition Zone Diameters (mm)a Klebsiella pneumoniae 24,50 ± 2,12 16.00 ± 1.41 11.78 ± 2.27 10.33 ± 2.31 Pseudomonas aeruginosa 9.00 ± 0.00 7.83 ± 0.76 7.50 ± 0.71 9.17 ± 0.29 Salmonella typhi 24.00 ± 1.41 12.22 ± 0.70 16.67 ± 1.15 10.50 ± 0.71 Escherichia coli 31.17 ± 2.84 13.88 ± 1.24 14.33 ± 0.47 10.75 ± 0.35 Bacillus cereus 19.33 ± 2.52 17.54 ± 0.30 17.00 ± 0.00 11.33 ± 1.53 Staphylococcus aureus 22.33 ± 1.15 8.33 ± 0.58 9.00 ± 0.00 9.22 ± 1.07 MRSA 29.00 ± 1.00 14.17 ± 1.18 9.83 ± 1.44 9.33 ± 1.15 Enterococcus faecalis 15.33 ± 1.04 8.33 ± 0.58 9.00 ± 1.41 9.00 ± 1.41 Candida albicans 19.67 ± 1.53 10.33 ± 2.31 10.33 ± 1.53 7.50 ± 0.71 a three repetitions average ± SD

Table 4b. Antimicrobial activity of the controls used by disc-diffusion diameter inhibition zones.

GM (10UI) AMC (10 g) OX1 (1 g) E15 (15 g)

Inhibition Zone Diameters (mm) Klebsiella pneumoniae 9 9 0 10 Pseudomonas aeruginosa 21 0 0 8 Salmonella typhi 21 30 0 16 Escherichia coli 31 24 0 15 Bacillus cereus 31 28 18 33 Staphylococcus aureus 22 32 24 29 MRSA 12 22 16 0 Enterococcus faecalis 23 25 0 29 GM: Gentamicine ; UI: unité internationale ; AMC: Amoxicilline – acide clavulanique ; OX1: Oxacilline ;E15: Erythromycine.

Table 4c. Antimicrobial activity of essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium by disc- diffusion diameter inhibition zones (values from literature).

Essential Oil Plant Thymus vulgaris Thymus algeriensis Mentha pulegium Morocco Morocco Libya Spain Algeria Tunisia Libya Iran Morocco Morocco Portugal Country [62] [63] [64] [38] [65] [66] [64] [54] [67] [68] [69]

Inhibition Zone Diameters

(mm)a Klebsiella - - - - - 13.5 - - - - - pneumoniae Pseudomonas - - - - - 14.5 - - - - - aeruginosa Salmonella - - - 36.0 - 15.0 - - - - 9.3 typhi Escherichia 0.33;1.33 34.7 - 19.6;28.3 - 14.0 - 21.8;23.7 12.6 2.3 Microbial coli strains Bacillus - - - - 0.0-22.0 30.0 - 16.0 - - - cereus Staphylococ- 1.33 35.0 45.0 0.0-14.0 - - 21.0 30.4 21.4 - cus aureus Enterococcus - - - - - 18.5 - - - - - faecalis Candida - - 20.0;40.0 - 9.0-18.66 - 5.0;10.0 16.0 - - - albicans 8 Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al.

Table 5a. MIC and MBC for the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium.

Thymus vulgaris Thymus algeriensis Mentha pulegium

(Djelfa) (Aflou-Laghouat) Microbial Strains MIC MBC MIC MBC (mg/mL) (mg/mL) MIC MBC MIC MBC (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL)

Klebsiella pneumoniae 1.016 2.032 2.114 > 4.227 2.030 > 4.059 2.070 2.070

Pseudomonas aeruginosa > 2.032 > 2.032 > 4.227 > 4.227 > 4.059 > 4.059 > 2.485 > 2.485

Salmonella typhi 0.508 1.016 2.114 3.044 3.004 4.059 1.035 2.070

Escherichia coli 0.508 1.016 3.044 4.227 3.004 > 4.059 1.035 1.035

Bacillus cereus 0.127 0.508 0.264 0.528 1.015 1.015 1.035 2.485

Staphylococcus aureus 1.016 1.016 1.057 1.057 1.015 1.015 1.035 2.070

MRSA 0.508 1.016 0.528 3.044 1.015 2.030 1.035 2.070

Enterococcus faecalis 0.127 1.016 0.528 1.057 0.507 1.015 1.035 2.070

Candida albicans 0.564 1.129 4.697 > 4.697 4.510 4.510 1.150 2.301

MIC: Minimum Inhibitory Concentration; MBC:Minimum Bactericide Concentration.

Table 5b. MIC (mg/mL) for the essential oil samples of Thymus vulgaris, Thymus algeriensis and Mentha pulegium (values from literature).

Plant Thymus vulgaris Thymus algeriensis Mentha pulegium

Libya Spain Iran Morocco Tunisia Libya Morocco Iran Morocco Morocco Portugal Country [64] [38] [70] [63] [66] [64] [71] [54] [67] [68] [69]

Klebsiella pneumoniae - - - - 6.0 ------

Pseudomonas aeruginosa 160.0 - - - 5.0 80.0 - - - - -

Salmonella typhi - < 0.2 - - 6.0 - - 4.0 - - 3.8

Microbial Escherichia coli - 0.5 - 9.3 6.0 - 2.0;4.0 4.0 4.0;2.0 1.0 3.2

strains Bacillus cereus - - - - 1.0 - - 1.0 - - -

Staphylococcus aureus 80.0 < 0.2 0.05 10.7 - 20.0 2.0 0.5 2.0 1.0 -

Enterococcus faecalis 80.0 0.05 - 3.0 80.0 - - - - -

Candida albicans ------2.0 - - - bly due to the difference in the compositions between the et al. [47] showed that the EOs containing phenolic struc- EOs. tures are very active against a wide spectrum of pathogenic fungi. The mechanism of toxicity of the phenols towards Soliman & Badiaa [43] tested the antifungal effect of fungi is based on the inactivation of fungal enzymes. Pheno- EOs of three species of Lamiaceae: T. vulgaris, M. viridis lic terpene also act by binding to the amino groups of the and Ocimum basilicum. They revealed that the three oils have inhibitory activities against the tested fungal species microbial membrane proteins causing the alteration of the permeability and leakage of intracellular components [25]. (Aspergillus flavus, A. parasiticus, A. ochraceus and This effect was also mentioned by Pinto et al. [48] by study- Fusarium moniliforme). For the same family Dambolena et ing the EO of T. pulegioides effect on the membrane of Can- al. [44] found that several samples of Ocimum basilicum and dida and Aspergillus. Ocimum gratissimum have an antifungal activity against Fusarium verticillioides. Ouraïni et al. [45] obtained signifi- 4. DISCUSSION cant antifungal activity for EOs of T. saturejoides and M. pulegium. The antifungal potency of the EO of thyme has 4.1. Essential Oil Yield and Chemical Composition also been shown by Yang & Clausen [46]. According to previous reports [49], several chemotypes The results show that the extracts with high phenol con- of T. vulgaris were determined: geraniol, -terpineol, sa- centration (T. vulgaris) have strong antifungal activities. binene hydrate (thuyanol), linalool, carvacrol, thymol and Several in vitro and in vivo studies conducted by Figueiredo 1,8-cineole. Several species belonging to Thymus genera Chemical Composition, Antioxidant and Antimicrobial Activities Current Nutrition & Food Science, 2017, Vol. 13, No. 0 9

Fig. (1). Two dimensional plot on axes F1 and F2 of the essential oil samples end their antioxidant activities using Principal Component Analysis. have similar compositions with a high content of phenolic founding of these In a matter of fact, the difference in com- terpenes (thymol or carvacrol) followed by a high percentage position of the EOs of T. algeriensis might be due to several of -terpinene and p-cymene: T. hyemalys [50] T. pulegioides factors, such as climate, season of harvest, stage of develop- [48], T. capitatus and T. herba-barona [28]. ment and even genetic profiles of the species [26, 50, 58-60]. Referring to literature, several major compounds were identified for the EO of this plant; previous studies confirm 4.2. Antioxidant Activity the predominance of pulegone as major compound: 73.40% The obtained results suggested that when the EOs of the on populations of Uruguay [51]; 85.40% on populations of studied plants exhibited high antioxidant activity in term of Morocco [52]; while others have identified piperitone and DPPH assay, they simultaneously exhibited low activity in piperitenone as the two dominant compounds [53, 54]. Ac- term of phosphomolebdenum assay; this finding could be cording to literature, three chemotypes have been estab- explained by the different reactions mechanisms involved in lished, pulegone type, piperitenone/piperitone type, and each assay and related to different active chemicals in each isomenthone / neoisomenthol type [55]. Beghidja et al. [56] case. studied the composition of several samples of pennyroyal from eastern Algeria, and claimed that these oils can be clas- 4.2.1. DPPH Assay sified into two chemotypes: one of pulegone (pulegone with The antioxidant activity of the EO of T. vulgaris is found a percentage between 52 and 87%), and a new chemotype poor in pulegone and rich of nonoxygenated terpenic frac- to be four times more active than the EO of T. algeriensis tions (-pinene, -thujene, -pinene, camphene, sabinene, - (Djelfa), and twelve times more active than the EO of M. phellandrene) and relatively high level of 1,8-cineol. pulegium. According to previous works which dealt with this plant Since the results of the investigated antioxidant activity from Algeria, it seems that there were some differences in showed a large variation in the values of EC50 and which the composition of the main compounds and which were were depending on the nature of the studied plant, it was related to the regions of collection [42, 57]. Indeed, Dob et mandatory to try to find a correlation between the chemical al. identified for a sample of T. algeriensis (Medea - Algeria) composition of the EOs and their antioxidant activity power the following major compounds: linalool (47.30%), thymol AAP (=1/EC50) values. This was carried out by applying (29.20%) and p-cymene (6.80%) [57]; While Hazzit et al. statistical methods involving Principal Component Analysis have suggested the existence of two chemotypes (samples PCA (Fig. 1). It was found that highest value of AAP (as it were collected from the Chrea National Park, and the Chlef was the case of T. vulgaris) was correlated with the previ- regions); the first was characterized by terpinyl acetate ously major identified components: p-cymene, thymol, li- (18.00%), trans-nerolidol (12.60%), -pinene (11.10%), nalool, thymol-ethyl-ether and -terpinene. At the opposite, borneol (9.00%) and bornyl acetate (7.70%), while the sec- T. algeriensis and M. pulegium presented the lowest activi- ond has presented the following major compounds: terpinen- ties, and where each correlated with their main EOs compo- 4-ol (10.60%), camphor (10.10%), p-cymene (9.90%), - nents, i.e. -terpinenyl acetate and pulegone, respectively. pinene (6.50%) and 1,8-cineole (6.50%) [42]. Comparing the Although, terpinen-4-ol and -terpineol were minor compo- 10 Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al.

Fig. (2). Two dimensional plot on axes F1 and F2 of the essential oil samples end their antimicrobial activity (disk diffusion test) using Prin- cipal Component Analysis. nents for T. algeriensis and M. pulegium, their relatively albicans. According to PCA analysis, this activity is due high percentages was also correlated with low antioxidant mainly to presence of thymol and thymol-methyl-ether. Al- activity. Finally, T. algeriensis from Aflou, had presented ternatively, the activity against Bacillus cereus was corre- practically no activity, and its EO was correlated with the lated with high percentages of camphor, borneol, - presence of simultaneously higher percentages of -pinene, terpinenyl acetate, terpinen-4-ol and -terpineol. Finally, the camphene, myrcene, eucalyptol, camphor and borneol. relatively slight improvement of activity of M. pulegium EO against Pseudomonas aeruginosa is probably due to the 4.2.2. Phosphomolybdenum Assay presence of the high percentage of pulegone. As a result, and First, let’s mention that vitamin E (antioxidant of refer- based on the PCA, it seems that pulegone does not contribute ence) which was used for comparison is almost 2 times less to any practical antimicrobial activity, since high percentage active than ascorbic acid (Table 3). Second, the values of the of pulegone imply slight improvement of this activity. antioxidant power for this assay were completely different to those found using DPPH assay. This time, T. algeriensis 4.3.1. Disc Diffusion Test (Aflou) and M. pulegium presented almost similar activities The analysis of the simultaneous values of MIC and MIB and were the most active EOs (6 to 7 times less active than with the chemical composition of the EOs against different ascorbic acid). At the opposite, T. vulgaris EO exhibited the bacteria using PCA revealed that the most active EOs plants lowest antioxidant power (almost 10 times less active than were those of T. algeriensis (Djelfa & Aflou) (Fig. 3). This ascorbic acid). strong activity was in strong correlation with several chemi- It was found that highest reduction power of the EOs of cals: eucalyptol, camphor, myrcene, -pinene, camphene, - T. algeriensis (Djelfa) is highly correlated with -terpinenyl terpineol, -terpinenyl acetate and terpinen-4-ol. For M. acetate, terpinen-4-ol and -terpineol. In another hand both pulegium where its EO presented relatively moderate activity T. algeriensis (Aflou) and M. pulegium with practically close (MBC) against Bacillus cereus, Staphylococcus aureus and reduction powers were correlated positively with different Enterococcus faecalis; this moderate activity is believed to components: -pinene, camphene, myrcene, eucalyptol, be correlated to high content of pulegone. camphor and borneol for T. algeriensis (Aflou); and only 4.3.2. Antifungal Activity pulegone for M. pulegium. At the contrary, T. vulgaris with the lowest reduction power was related to higher percentages It was found that the EOs were exhibiting high MIC and of p-cymene, thymol, linalool, thymol-ethyl-ether and - at the same time presenting low or undetected activities in terpinene. term of MIB values. The results of PCA showed (Fig. 4), that the highest MIC 4.3. Antimicrobial Activity values were correlated for different sets of EOs components According to Fig. 2, the T. vulgaris was found to be the for both T. algeriensis (Djelfa) and M. pulegium. Moreover, most active EO and exhibited important resistance against for almost the same components observed previously with most of studied bacteria: Staphylococcus aureus, Escherichia disc diffusion test, the high MIC value for M. pulegium was coli, Salmonella typhi, Klebsiella pneumoniae and Candida attributed to the high composition of pulegone, but for T. Chemical Composition, Antioxidant and Antimicrobial Activities Current Nutrition & Food Science, 2017, Vol. 13, No. 0 11

Fig. (3). Two dimensional plot on axes F1 and F2 of the essential oil samples end their antimicrobial activity (minimal inhibitory and bactericidal concentrations) using Principal Component Analysis.

Fig. (4). Two dimensional plot on axes F1 and F2 of the essential oil samples end their antifungal activity using Principal Component Analysis. algeriensis (Djelfa) it was due to the presence of eucalyptol, CONCLUSION -terpinenyl acetate, terpinen-4-ol, -terpineol, -pinene, As a conclusion, the EOs tested in this study showed borneol and camphene. The EO of T. vulgaris with the low- moderate to strong antioxidant and antimicrobial activities. est value of MIC was correlated with the following compo- Almost all EOs exhibited a radical scavenging activity nents: thymol-ethyl-ether, -terpinene, thymol, p-cymene and (DPPH assay), with different values of EC . T. vulgaris EO linalool. At the opposite, the EO of this later plant presented 50 had the lowest value of EC which makes it the strongest a good MFC value in comparison with the rest of the studied 50 plants, in which they did not offer any measurable activity in EO among the tested samples. For antimicrobial activity, all the samples had inhibited the growth of the tested microbial the range of the studied concentrations. This high activity is strains, with the strongest antibacterial and antifungal activ- due to the presence of thymol-ethyl-ether, -terpinene, thy- ity attributed to T. vulgaris EO. The study of the relation mol, p-cymene and linalool. 12 Current Nutrition & Food Science, 2017, Vol. 13, No. 0 Benabed et al. between the chemical composition of the EOs and the anti- induced dermatomycoses. Drug Dev Ind Pharm 2008; 34(12): oxidant or the antimicrobial activity revealed the presence of 1388-93. [15] Sokovi MD, Vukojevi J, Marin PD, et al. Chemical composition different strong correlations with some major identified of essential oils of Thymus and Mentha species and their antifungal compounds in each case of study. It could be concluded that, activities. Molecules 2009; 14(1): 238-49. thyme, T. vulgaris EO were effective as antioxidant activity [16] Baba-Aissa F. Encyclopédie des plantes utiles: Flore méditerra- due, in part, to the presence of several compounds, like thy- néenne (Maghreb,Europe méridionale) Substances végétales mol, thymol-methyl-ether, linalool and carverol, in their d'Afrique , d'Orient dt d'Occident: Alger : El Maarifa; 2011. [17] Nikoli M, Glamo lija J, Ferreira IC, et al. Chemical composition, chemical compositions. They could be used as flavoring antimicrobial, antioxidant and antitumor activity of Thymus serpyl- agents and good sources of antioxidants in making food with lum L., Thymus algeriensis Boiss. and Reut and Thymus vulgaris L. healthy benefits and good sensory acceptability. Further essential oils. Ind Crops Prod 2014; 52: 183-90. studies are required to collect more data about the toxicity of [18] Beloued A. Plantes médicinales d'Algérie: Alger: Offices des publications universitaires; 2001. these EOs. [19] Boukhebti H, Chaker AN, Belhadj H, et al. Chemical composition and antibacterial activity of Mentha pulegium L. and Mentha spi- CONFLICT OF INTEREST cata L. essential oils. Der Pharmacia Lett 2011; 3(4): 267-75. [20] Adams RP. Identification of essential oil components by gas chro- The authors confirm that this article content has no con- matography/ mass spectrometry, 4th ed. Allured Publishing Corpo- flict of interest. ration: Illinois 2007; pp. 804. [21] Molyneux P. The use of the stable free radical diphenylpicrylhy- drazyl (DPPH) for estimating antioxidant activity. Songklanakarin ACKNOWLEDGEMENTS J Sci Technol 2004; 26(2): 211-9. [22] Prietto P, Pineda M, Aquilar M. Spectrophotometric quantification The authors wish to thank the Algerian Ministry of High of antioxidant capacity through the formation of phosphomolybde- Education and Scientific Research for funding the Research num complex: Specification application to the determination of vi- Laboratory of Fundamental Sciences-Laghouat, Algeria. tamin E. Anal Biochem 1999; 269: 337-41. [23] De Billerbeck V-G. Huiles essentielles et bactéries résistantes aux antibiotiques. Phytothérapie 2007; 5(5): 249-53. REFERENCES [24] Cao L, Si JY, Liu Y, et al. Essential oil composition, antimicrobial and antioxidant properties of Mosla chinensis Maxim. Food Chem [1] Gurib-Fakim A. Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspects Med 2006; 27: 1-93. 2009; 115(3): 801-5. [25] El Ajjouri M, Satrani B, Ghanmi M, et al. Activité antifongique des [2] Nostro A, Germano M, D’angelo V, et al. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial ac- huiles essentielles de Thymus bleicherianus Pomel et Thymus capitatus (L.) Hoffm. & Link contre les champignons de pourriture du tivity. Lett Appl Microbiol 2000; 30(5): 379-84. [3] Kulisic T, Radonic A, Katalinic V, et al. Use of different methods bois d'oeuvre. Biotechnologie Agronomie Société Et Environ 2008; 12(4): 345-51. for testing antioxidative activity of oregano essential oil. Food Chem 2004; 85(4): 633-40. [26] Amarti F, Satrani B, Ghanmi M, et al. Composition chimique et activité antimicrobienne des huiles essentielles de Thymus alge- [4] Chouliara E, Karatapanis A, Savvaidis I, et al. Combined effect of oregano essential oil and modified atmosphere packaging on shelf- riensis Boiss. & Reut. et Thymus ciliatus (Desf.) Benth. du Maroc. Biotechnologie Agronomie Société Et Environ 2010; 14(1): 141-8. life extension of fresh chicken breast meat, stored at 4 °C. Food Microbiol 2007; 24(6): 607-17. [27] Zouari N, Fakhfakh N, Zouari S, et al. Chemical composition, angiotensin I-converting enzyme inhibitory, antioxidant and antim- [5] Gómez-Estaca J, de Lacey AL, López-Caballero M, et al. Biode- gradable gelatin-chitosan films incorporated with essential oils as icrobial activities of essential oil of Tunisian Thymus algeriensis Boiss. et Reut.(Lamiaceae). Food Bioprod Process 2011; 89(4): antimicrobial agents for fish preservation. Food Microbiol 2010; 27(7): 889-96. 257-65. [28] Cosentino S, Tuberoso C, Pisano B, et al. Invitro antimicrobial [6] Holley RA, Patel D. Improvement in shelf-life and safety of perish- able foods by plant essential oils and smoke antimicrobials. Food activity and chemical composition of Sardinian thymus essential oils. Lett Appl Microbiol 1999; 29(2): 130-5. Microbiol 2005; 22(4): 273-92. [7] Silva LF, das Graças Cardoso M, Batista LR, et al. Chemical char- [29] Bouhdid S, Idaomar M, Zhiri A, et al., editors. Thymus essential oils: Chemical composition and in vitro antioxidant and antibacte- acterization, antibacterial and antioxidant activities of essential oils of Mentha viridis L. and Mentha pulegium L. (L). Am J Plant Sci rial activities. Congrès International de Biochimie: Biochimie, Substances Naturelles et Environnement 2006 9-12 ; Agadir, Mo- 2015; 6(5): 666. [8] Grigore A, Paraschiv I, Colceru-Mihul S, et al. Chemical composi- rocco. [30] Hussain AI, Anwar F, Nigam PS, et al. Antibacterial activity of tion and antioxidant activity of Thymus vulgaris L. volatile oil obtained by two different methods. Rom Biotechnol Lett 2010; 15(4): some Lamiaceae essential oils using resazurin as an indicator of cell growth. LWT-Food Sci Technol 2011; 44(4): 1199-206. 5436-43. [9] Ramadan KM, Ashoush I, El-Batawy O. Comparative evaluation of [31] Carson C, Riley T. Antimicrobial activity of the major components of the essential oil of Melaleuca alternifolia. J Appl Bacteriol three essential oils as functional antioxidants and natural flavoring agents in ice cream. World Appl Sci J 2013; 23(2): 159-66. 1995; 78(3): 264-9. [32] Klan nik A, Piskernik S, Jerek B, et al. Evaluation of diffusion [10] Rustaiyan A, Masoudi S, Monfared A, et al. Volatile constituents of three Thymus species grown wild in Iran. Planta Med 2000; and dilution methods to determine the antibacterial activity of plant extracts. J Microbiol Methods 2010; 81(2): 121-6. 66(02): 197-8. [11] Marino M, Bersani C, Comi G. Antimicrobial activity of the essen- [33] Imelouane B, Amhamdi H, Wathelet JP, et al. Chemical composition and antimicrobial activity of essential oil of thyme (Thymus tial oils of Thymus vulgaris L. measured using a bioimpedometric method. J Food Prot 1999; 62(9): 1017-23. vulgaris) from Eastern Morocco. Int J Agric Biol 2009; 11(2): 205- 8. [12] PinaVaz C, Gonçalves Rodrigues A, Pinto E, et al. Antifungal activity of Thymus oils and their major compounds. J Eur Acad [34] Kaloustian J, Chevalier J, Mikail C, et al. Étude de six huiles essentielles: Composition chimique et activité antibactérienne. Phy- Dermatol Venereol 2004; 18(1): 73-8. [13] Simandi B, Hajdu V, Peredi K, et al. Antioxidant activity of pi- tothérapie 2008; 6(3): 160-4. [35] Govaris A, Botsoglou E, Sergelidis D, et al. Antibacterial activity lotplant alcoholic and supercritical carbon dioxide extracts of thyme. Eur J Lipid Sci Technol 2001; 103(6): 355-8. of oregano and thyme essential oils against Listeria monocytogenes and Escherichia coli O157: H7 in feta cheese packaged under [14] Sokovi M, Glamo lija J, iri A, et al. Antifungal activity of the essential oil of Thymus vulgaris L. and thymol on experimentally modified atmosphere. LWT-Food sci technol 2011; 44(4): 1240-4. Chemical Composition, Antioxidant and Antimicrobial Activities Current Nutrition & Food Science, 2017, Vol. 13, No. 0 13

[36] Kabouche Z, Boutaghane N, Laggoune S, et al. Comparative anti- [55] Stoyanova A, Georgiev E, Kula J, et al. Chemical composition of bacterial activity of five Lamiaceae essential oils from Algeria. Int the essential oil of Mentha pulegium L. from Bulgaria. J Essent Oil J Aromatherapy 2005; 15(3): 129-33. Res 2005; 17(5): 475-6. [37] Naeini A, Khosravi A, Chitsaz M, et al. Anti-Candida albicans [56] Beghidja N, Bouslimani N, Benayache F, et al. Composition of the activity of some Iranian plants used in traditional medicine. J Med oils from Mentha pulegium grown in different areas of the East of Mycol 2009; 19(3): 168-72. Algeria. Chem Nat Compd 2007; 43(4): 481-3. [38] Rota MC, Herrera A, Martínez RM, et al. Antimicrobial activity [57] Dob T, Dahmane D, Benabdelkader T, et al. Studies on the essen- and chemical composition of Thymus vulgaris, Thymus zygis and tial oil composition and antimicrobial activity of Thymus algerien- Thymus hyemalis essential oils. Food Control 2008; 19(7): 681-7. sis Boiss. et Reut. Int J Aromatherapy 2006; 16(2): 95-100. [39] Saad A, Fadli M, Bouaziz M, et al. Anticandidal activity of the [58] nan M, Kirpik M, Kaya DA, et al. Effect of harvest time on essen- essential oils of Thymus maroccanus and Thymus broussonetii and tial oil composition of Thymbra spicata L. growing in flora of their synergism with amphotericin B and fluconazol. Phytomedi- Adıyaman. Adv Environ Biol 2011; 5(2): 356-8. cine 2010; 17(13): 1057-60. [59] Sellami IH, Maamouri E, Chahed T, et al. Effect of growth stage [40] Smith-Palmer A, Stewart J, Fyfe L. Antimicrobial properties of on the content and composition of the essential oil and phenolic plant essential oils and essences against five important food-borne fraction of sweet marjoram (Origanum majorana L.). Ind Crops pathogens. Lett Appl Microbiol 1998; 26(2): 118-22. Prod 2009; 30(3): 395-402. [41] Tohidpour A, Sattari M, Omidbaigi R, et al. Antibacterial effect of [60] Telci I, Demirtas I, Bayram E, et al. Environmental variation on essential oils from two medicinal plants against Methicillin- aroma components of pulegone/piperitone rich spearmint (Mentha resistant Staphylococcus aureus (MRSA). Phytomedicine 2010; spicata L.). Ind Crops Prod 2010; 32(3): 588-92. 17(2): 142-5. [61] Senatore F, Urrunaga Soria E, Urrunaga Soria R, et al. Essential oil [42] Hazzit M, Baaliouamer A, Veríssimo A, et al. Chemical composi- of Eremocharis triradiata (Wolff.) Johnston (Apiaceae) growing tion and biological activities of Algerian Thymus oils. Food Chem wild in Peru. Flavour Fragrance J 1997; 12(4): 257-9. 2009; 116(3): 714-21. [62] Imelouane B, Amhamdi H, Wathelet J-P, et al. Chemical composi- [43] Soliman K, Badeaa R. Effect of oil extracted from some medicinal tion and antimicrobial activity of essential oil of thyme (Thymus plants on different mycotoxigenic fungi. Food Chem Toxicol 2002; vulgaris) from Eastern Morocco. Int J Agric Biol 2009; 11(2): 205- 40(11): 1669-75. 8. [44] Dambolena JS, Zunino MP, López AG, et al. Essential oils compo- [63] Zantar S, Haouzi R, Chabbi M, et al. Effect of gamma irradiation sition of Ocimum basilicum L. and Ocimum gratissimum L. from on chemical composition, antimicrobial and antioxidant activities Kenya and their inhibitory effects on growth and fumonisin pro- of Thymus vulgaris and Mentha pulegium essential oils. Radiat duction by Fusarium verticillioides. Innov Food Sci Emerg Tech- Phys Chem 2015; 115(0): 6-11. nol 2010; 11(2): 410-4. [64] Nikoli M, Glamolija J, Ferreira ICFR, et al. Chemical composi- [45] Ouraïni D, Agoumi A, Ismaïli-Alaoui M, et al. Activité antifongi- tion, antimicrobial, antioxidant and antitumor activity of Thymus que de l’acide oléique et des huiles essentielles de Thymus sature- serpyllum L., Thymus algeriensis Boiss. and Reut and Thymus vul- joides L. et de Mentha pulegium L., comparée aux antifongiques garis L. essential oils. Ind Crops Prod 2014; 52(0): 183-90. dans les dermatoses mycosiques. Phytothérapie 2007; 5(1): 6-14. [65] Hazzit M, Baaliouamer A, Veríssimo AR, et al. Chemical composi- [46] Yang VW, Clausen CA. Antifungal effect of essential oils on tion and biological activities of Algerian Thymus oils. Food Chem southern yellow pine. Int Biodeterior Biodegrad 2007; 59(4): 302- 2009; 116(3): 714-21. 6. [66] Zouari N, Fakhfakh N, Zouari S, et al. Chemical composition, [47] Figueiredo A, Barroso J, Pedro L, et al. Portuguese Thymbra and angiotensin I-converting enzyme inhibitory, antioxidant and antim- Thymus species volatiles: chemical composition and biological ac- icrobial activities of essential oil of Tunisian Thymus algeriensis tivities. Curr Pharm Des 2008; 14(29): 3120-40. Boiss. et Reut. (Lamiaceae). Food Bioprod Process 2011; 89(4): [48] Pinto E, Pina-Vaz C, Salgueiro L, et al. Antifungal activity of the 257-65. essential oil of Thymus pulegioides on Candida, Aspergillus and [67] Cherrat L, Espina L, Bakkali M, et al. Chemical composition, dermatophyte species. J Med Microbiol 2006; 55(10): 1367-73. antioxidant and antimicrobial properties of Mentha pulegium, [49] KeefoverRing K, Thompson JD, Linhart YB. Beyond six scents: Lavandula stoechas and Satureja calamintha Scheele essential oils Defining a seventh Thymus vulgaris chemotype new to southern and an evaluation of their bactericidal effect in combined proc- France by ethanol extraction. Flavour Fragrance J 2009; 24(3): esses. Innov Food Sci Emerg Technol 2014; 22(0): 221-9. 117-22. [68] Ait-Ouazzou A, Lorán S, Arakrak A, et al. Evaluation of the [50] Jordán M, Martínez R, Goodner K, et al. Seasonal variation of chemical composition and antimicrobial activity of Mentha pule- Thymus hyemalis Lange and Spanish Thymus vulgaris L. essential gium, Juniperus phoenicea, and Cyperus longus essential oils from oils composition. Ind Crops Prod 2006; 24(3): 253-63. Morocco. Food Res Int 2012; 45(1): 313-9. [51] Lorenzo D, Paz D, Dellacassa E, et al. Essential oils of Mentha [69] Teixeira B, Marques A, Ramos C, et al. European pennyroyal pulegium and Mentha rotundifolia from Uruguay. Braz Arch Biol (Mentha pulegium) from Portugal: Chemical composition of essen- Technol 2002; 45(4): 519-24. tial oil and antioxidant and antimicrobial properties of extracts and [52] Bouchra C, Achouri M, Hassani LI, et al. Chemical composition essential oil. Ind Crops Prod 2012; 36(1): 81-7. and antifungal activity of essential oils of seven Moroccan Labiatae [70] Nezhadali A, Nabavi M, Rajabian M, et al. Chemical variation of against Botrytis cinerea Pers: Fr. J Ethnopharmacol 2003; 89(1): leaf essential oil at different stages of plant growth and in vitro an- 165-9. tibacterial activity of Thymus vulgaris Lamiaceae, from Iran. Beni- [53] Derwich E, Benziane Z, Taouil R, et al. Comparative essential oil Suef Univ J Basic Appl Sci 2014; 3(2): 87-92. composition of leaves of Mentha rotundifolia and Mentha pulegium [71] Cherrat L, Espina L, Bakkali M, et al. Chemical composition, a traditional herbal medicine in Morocco. American-Eurasian J antioxidant and antimicrobial properties of Mentha pulegium, Sustain Agric 2010; 4(1): 47-54. Lavandula stoechas and Satureja calamintha Scheele essential oils [54] Mahboubi M, Haghi G. Antimicrobial activity and chemical com- and an evaluation of their bactericidal effect in combined proc- position of Mentha pulegium L. essential oil. J Ethnopharmacol esses. Innov Food Sci Emerg Technol 2014; 22: 221-9. 2008; 119(2): 325-7.

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